Leucine-based motifs and enhanced biological persistence of clostridial neurotoxins

ABSTRACT

Modified neurotoxin comprising neurotoxin including structural modification, wherein the structural modification alters the biological persistence, such as the biological half-life and/or a biological activity of the modified neurotoxin relative to an identical neurotoxin without the structural modification. In one embodiment, methods of making the modified neurotoxin include using recombinant techniques. In another embodiment, methods of using the modified neurotoxin to treat conditions include treating various disorders, neuromuscular aliments and pain.

CROSS REFERENCE

This application is a continuation in part of application Ser. No.09/620,840, filed Jul. 21, 2000.

BACKGROUND

The present invention relates to modified neurotoxins, particularlymodified Clostridial neurotoxins, and use thereof to treat variousconditions including conditions that have been treated using naturallyoccurring botulinum toxins.

Botulinum toxin, for example, botulinum toxin type A, has been used inthe treatment of numerous conditions including pain, skeletal muscleconditions, smooth muscle conditions and glandular conditions. Botulinumtoxins are also used for cosmetic purposes.

Numerous examples exist for treatment using botulinum toxin. Forexamples of treating pain see Aoki, et al., U.S. Pat. No. 6,113,915 andAoki, et al., U.S. Pat. No. 5,721,215. For an example of treating aneuromuscular disorder, see U.S. Pat. No. 5,053,005, which suggeststreating curvature of the juvenile spine, i.e., scoliosis, with anacetylcholine release inhibitor, preferably botulinum toxin A. For thetreatment of strabismus with botulinum toxin type A, see Elston, J. S.,et al., British Journal of Ophthalmology, 1985, 69, 718-724 and 891-896.For the treatment of blepharospasm with botulinum toxin type A, seeAdenis, J. P., et al., J. Fr. Ophthalmol., 1990, 13 (5) at pages259-264. For treating spasmodic and oromandibular dystonia torticollis,see Jankovic et al., Neurology, 1987, 37, 616-623. Spasmodic dysphoniahas also been treated with botulinum toxin type A. See Blitzer et al.,Ann. 0 to 1. Rhino. Laryngol, 1985, 94, 591-594. Lingual dystonia wastreated with botulinum toxin type A according to Brin et al., Adv.Neurol. (1987) 50, 599-608. Cohen et al., Neurology (1987) 37 (Suppl.1), 123-4, discloses the treatment of writer's cramp with botulinumtoxin type A.

It would be beneficial to have botulinum toxins with altered biologicalpersistence and/or altered biological activity. For example, a botulinumtoxin can be used to immobilize muscles and prevent limb movements aftertendon surgery to facilitate recovery. It would be beneficial to have abotulinum toxin (such as a botulinum toxin type A) which exhibits areduced period of biological persistence so that a patient can regainmuscle use and mobility at about the time they recover from surgery.Furthermore, a botulinum toxin with an altered biological activity, suchas an enhanced biological activity can have utility as a more efficienttoxin (i.e. more potent per unit amount of toxin), so that less toxincan be used.

Additionally, there is a need for modified neurotoxins (such as modifiedClostridial toxins) which can exhibit an enhanced period of biologicalpersistence and modified neurotoxins (such as modified Clostridialtoxins) with reduced biological persistence and/or biological activityand methods for preparing such toxins.

Definitions

Before proceeding to describe the present invention, the followingdefinitions are provided and apply herein.

“Heavy chain” means the heavy chain of a Clostridial neurotoxin. It hasa molecular weight of about 100 kDa and can be referred to herein asHeavy chain or as H.

“H_(N)” means a fragment (having a molecular weight of about 50 kDa)derived from the Heavy chain of a Clostridial neurotoxin which isapproximately equivalent to the amino terminal segment of the Heavychain, or the portion corresponding to that fragment in the intact Heavychain. It is believed to contain the portion of the natural or wild typeClostridial neurotoxin involved in the translocation of the light chainacross an intracellular endosomal membrane.

“H_(C)” means a fragment (about 50 kDa) derived from the Heavy chain ofa Clostridial neurotoxin which is approximately equivalent to thecarboxyl terminal segment of the Heavy chain, or the portioncorresponding to that fragment in the intact Heavy chain. It is believedto be immunogenic and to contain the portion of the natural or wild typeClostridial neurotoxin involved in high affinity binding to variousneurons (including motor neurons), and other types of target cells.

“Light chain” means the light chain of a Clostridial neurotoxin. It hasa molecular weight of about 50 kDa, and can be referred to as lightchain, L or as the proteolytic domain (amino acid sequence) of aClostridial neurotoxin. The light chain is believed to be effective asan inhibitor of exocytosis, including as an inhibitor ofneurotransmitter (i.e. acetylcholine) release when the light chain ispresent in the cytoplasm of a target cell.

“Neurotoxin” means a molecule that is capable of interfering with thefunctions of a cell, including a neuron. The “neurotoxin” can benaturally occurring or man-made. The interfered with function can beexocytosis.

“Modified neurotoxin” means a neurotoxin which includes a structuralmodification. In other words, a “modified neurotoxin” is a neurotoxinwhich has been modified by a structural modification. The structuralmodification changes the biological persistence, such as the biologicalhalf-life (i.e. the duration of action of the neurotoxin) and/or thebiological activity of the modified neurotoxin relative to theneurotoxin from which the modified neurotoxin is made or derived. Themodified neurotoxin is structurally different from a naturally existingneurotoxin.

“Mutation” means a structural modification of a naturally occurringprotein or nucleic acid sequence. For example, in the case of nucleicacid mutations, a mutation can be a deletion, addition or substitutionof one or more nucleotides in the DNA sequence. In the case of a proteinsequence mutation, the mutation can be a deletion, addition orsubstitution of one or more amino acids in a protein sequence. Forexample, a specific amino acid comprising a protein sequence can besubstituted for another amino acid, for example, an amino acid selectedfrom a group which includes the amino acids alanine, aspargine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, tyrosine or any othernatural or non-naturally occurring amino acid or chemically modifiedamino acids. Mutations to a protein sequence can be the result ofmutations to DNA sequences that when transcribed, and the resulting mRNAtranslated, produce the mutated protein sequence. Mutations to a proteinsequence can also be created by fusing a peptide sequence containing thedesired mutation to a desired protein sequence.

“Structural modification” means any change to a neurotoxin that makes itphysically or chemically different from an identical neurotoxin withoutthe structural modification.

“Biological persistence” or “persistence” means the time duration ofinterference or influence caused by a neurotoxin or a modifiedneurotoxin with a cellular (such as a neuronal) function, including thetemporal duration of an inhibition of exocytosis (such as exocytosis ofneurotransmitter, for example, acetylcholine) from a cell, such as aneuron.

“Biological half-life” or “half-life” means the time that theconcentration of a neurotoxin or a modified neurotoxin, preferably theactive portion of the neurotoxin or modified neurotoxin, for example,the light chain of Clostridial toxins, is reduced to half of theoriginal concentration in a mammalian cell, such as in a mammalianneuron.

“Biological activity” or “activity” means the amount of cellularexocytosis inhibited from a cell per unit of time, such as exocytosis ofa neurotransmitter from a neuron.

“Target cell” means a cell (including a neuron) with a binding affinityfor a neurotoxin or for a modified neurotoxin.

SUMMARY

New structurally modified neurotoxins have been discovered. The presentstructurally modified neurotoxins can provide substantial benefits, forexample, enhanced or decreased biological persistence and/or biologicalhalf-life and/or enhanced or decreased biological activity as comparedto the unmodified neurotoxin.

In accordance with the present invention, there are providedstructurally modified neurotoxins, which include a neurotoxin and astructural modification. The structural modification is effective toalter a biological persistence of the structurally modified neurotoxinrelative to an identical neurotoxin without the structural modification.Also, the structurally modified neurotoxin is structurally differentfrom a naturally existing neurotoxin.

The present invention also encompasses a modified neurotoxin comprisinga neurotoxin with a structural modification, wherein said structuralmodification is effective to alter a biological activity of saidmodified neurotoxin relative to an identical neurotoxin without saidstructural modification, and wherein said modified neurotoxin isstructurally different from a naturally existing neurotoxin. Thisstructural modification can be effective to reduce an exocytosis from atarget cell by more than the amount of the exocytosis reduced from thetarget cell by an identical neurotoxin without said structuralmodification. Alternately, the structural modification can be effectiveto reduce an exocytosis from a target cell by less than the amount ofthe exocytosis reduced from the cell by an identical neurotoxin withoutsaid structural modification. Significantly, the exocytosis can beexocytosis of a neurotransmitter and the modified neurotoxin can exhibitan altered biological activity without exhibiting an altered biologicalpersistence. The structural modification can comprise a leucine-basedmotif. Additionally, the modified neurotoxin can exhibits an alteredbiological activity as well as an altered biological persistence. Thepresent invention also includes the circumstances where: (a) themodified neurotoxin exhibits an increased biological activity as well asan increased biological persistence; (b) the modified neurotoxinexhibits an increased biological activity and a reduced biologicalpersistence; (c) the modified neurotoxin exhibits a decreased biologicalactivity and a decreased biological persistence, and; (d) the modifiedneurotoxin exhibits an decreased biological activity and an increasedbiological persistence.

Importantly, a unit amount (i.e. on a molar basis) of the modifiedneurotoxin can be more efficient to reduce an exocytosis from a cellthan is a unit amount of the naturally existing neurotoxin. In otherwords, a unit amount of a modified neurotoxin, such as a modifiedbotulinum toxin type A, can cleave its' intracellular substrate (SNAP)in a manner such that a greater inhibition of neurotransmitterexocytosis results (i.e. less neurotransmitter is released from thecell), as compared to the inhibition of neurotransmitter exocytosisexhibited by the naturally occurring neurotoxin.

Further in accordance with the present invention, are structurallymodified neurotoxins, wherein a structural modification is effective toenhance a biological persistence of the modified neurotoxin. Theenhanced biological persistence of the structurally modified neurotoxincan be due, at least in part, to an increased half-life and/orbiological activity of the structurally modified neurotoxin.

Still further in accordance with the present invention, there areprovided structurally modified neurotoxins wherein a biologicalpersistence of the structurally modified neurotoxin is reduced relativeto that of an identical neurotoxin without the structural modification.This reduction in biological persistence can be due, at least in part,to a decreased biological half-life and/or activity of the structurallymodified neurotoxins.

Still further in accordance with the present invention, there areprovided structurally modified neurotoxins wherein the structuralmodification comprises a number of amino acids. For example, the numberof amino acids comprising the structural modification can be 1 or moreamino acids, from 1 to about 22 amino acids, from 2 to about 10 aminoacids, and from about 4 to about 7 amino acids.

In one embodiment, the structural modifications of the structurallymodified neurotoxins can comprise an amino acid. The amino acid cancomprise an R group containing a number of carbons. For example, thenumber of carbon atoms in the amino acid can be 1 or more, from 1 toabout 20 carbons, from 1 to about 12 carbons, from 1 to about 9 carbons,from 2 to about 6 carbons, and about 4 carbons. R group as used in thisapplication refers to amino acid side chains. For example, the R groupfor alanine is CH₃, and, for example, the R group for serine is CH₂OH.

In another embodiment, there are provided structurally modifiedneurotoxins wherein the modification comprises an amino acid. The aminoacid can comprise an R group which is substantially hydrocarbyl.

In still another embodiment, there are provided structurally modifiedneurotoxins wherein the structural modification comprises an amino acid.The amino acid further can comprise an R group that includes at leastone heteroatom.

Further in accordance with the present invention, there are providedstructurally modified neurotoxins wherein the structural modificationcomprises, for example, a leucine-based motif, a tyrosine-based motif,and/or an amino acid derivative. Examples of an amino acid derivativethat can comprise a structurally modified neurotoxin ar a myristylatedamino acid, an N-glycosylated amino acid, and a phosphorylated aminoacid. The phosphorylated amino acids can be phosphorylated by, forexample, casein kinase II, protein kinase C, and tyrosine kinase.

Still further in accordance with the present invention, there areprovided structurally modified neurotoxins which can include astructural modification. The neurotoxin can comprise three amino acidsequence regions. The first region can be effective as a cellularbinding moiety. This binding moiety can be a binding moiety for a targetcell, such as a neuron. The binding moiety can be the carboxyl terminusof a botulinum toxin heavy chain. It is well known that the carboxylterminus of a botulinum toxin heavy chain can be effective to bind, forexample, receptors found on certain cells, including certain nervecells. In one embodiment, the carboxyl terminus binds to receptors foundon a presynaptic membrane of a nerve cell. The second region can beeffective to translocate a structurally modified neurotoxin, or a partof a structurally modified neurotoxin across an endosome membrane. Thethird region can be effective to inhibit exocytosis from a target cell.The inhibition of exocytosis can be inhibition of neurotransmitterrelease, such as acetylcholine from a presynaptic membrane. For example,it is well known that the botulinum toxin light chain is effective toinhibit, for example, acetylcholine (as well as other neurotransmitters)release from various neuronal and non-neuronal cells.

At least one of the first, second or third regions can be substantiallyderived from a Clostridial neurotoxin. The third region can include thestructural modification. In addition, the modified neurotoxin can bestructurally different from a naturally existing neurotoxin. Also, thestructural modification can be effective to alter a biologicalpersistence of the modified neurotoxin relative to an identicalneurotoxin without the structural modification.

In one embodiment, there are provided structurally modified neurotoxins,wherein the neurotoxin can be botulinum serotype A, B, C₁, C₂, D, E, F,G, tetanus toxin and/or mixtures thereof.

In another embodiment, there are provided structurally modifiedneurotoxins where the third region can be derived from botulinum toxinserotype A. In addition, there are provided structurally modifiedneurotoxins wherein the third region can not be derived from botulinumserotype A.

In still another embodiment, there are provided structurally modifiedneurotoxins wherein the structural modification includes a biologicalpersistence enhancing component effective to enhance the biologicalpersistence of the structurally modified neurotoxin. The enhancing ofthe biological persistence can be at least in part due to an increase inbiological half-life and/or activity of the structurally modifiedneurotoxin.

Further in accordance with the present invention, there are providedstructurally modified neurotoxins comprising a biological persistenceenhancing component, wherein the biological persistence enhancingcomponent can comprise a leucine-based motif. The leucine-based motifcan comprise a run of 7 amino acids, where a quintet of amino acids anda duplet of amino acids can comprise the leucine-based motif. Thequintet of amino acids can define the amino terminal end of theleucine-based motif. The duplet of amino acids can define the carboxylend of the leucine-based motif. There are provided structurally modifiedneurotoxins wherein the quintet of amino acids can comprise one or moreacidic amino acids. For example, the acidic amino acid can be glutamateor aspartate. The quintet of amino acids can comprise a hydroxylcontaining amino acid. The hydroxyl containing amino acid can be, forexample, a serine, a threonine or a tyrosine. This hydroxyl containingamino acid can be phosphorylated. At least one amino acid comprising theduplet of amino acids can be a leucine, isoleucine, methionine, alanine,phenylalanine, tryptophan, valine or tyrosine. In addition, the dupletof amino acids in the leucine-based motif can be leucine-leucine,leucine-isoleucine, isoleucine-leucine or isoleucine-isoleucine,leucine-methionine. The leucine-based motif can be an amino acidsequence ofphenylalanine-glutamate-phenylalanine-tyrosine-lysine-leucine-leucine.

In one embodiment, there are provided structurally modified neurotoxinswherein the modification can be a tyrosine-based motif. Thetyrosine-based motif can comprise four amino acids. The amino acid atthe N-terminal end of the tyrosine-based motif can b a tyrosine. Theamino acid at the C-terminal end of the tyrosine-based motif can be ahydrophobic amino acid.

Further in accordance with the present invention, the third region canbe derived from botulinum toxin serotype A or form one of the otherbotulinum toxin serotypes.

Still further in accordance with the present invention, there areprovided structurally modified neurotoxins where the biologicalpersistence of the structurally modified neurotoxin can be reducedrelative to an identical neurotoxin without the structural modification.The reduced biological persistence can be in part due a decreasedbiological half-life and/or to a decrease biological activity of theneurotoxin.

In one embodiment, there are provided structurally modified neurotoxins,where the structural modification can include a leucine-based motif witha mutation of one or more amino acids comprising the leucine-basedmotif. The mutation can be a deletion or substitution of one or moreamino acids of the leucine-based motif.

In another embodiment, there are provided structurally modifiedneurotoxins, where the structural modification includes a tyrosine-basedmotif with a mutation of one or more amino acids comprising thetyrosine-based motif. For example, the mutation can be a deletion orsubstitution of one or more amino acids of the tyrosine-based motif.

In still another embodiment, there are provided structurally modifiedneurotoxins, wherein the structural modification comprises an amino acidderivative with a mutation of the amino acid derivative or a mutation toa nucleotide or amino acid sequence which codes for the derivativizationof the amino acid. For example, a deletion or substitution of thederivatized amino acid or a nucleotide or amino acid sequenceresponsible for a derivatization of the derivatized amino acid. Theamino acid derivative can be, for example, a myristylated amino acid, anN-glycosylated amino acid, or a phosphorylated amino acid. Thephosphorylated amino acid can be produced by, for example, casein kinaseII, protein kinase C or tyrosine kinase.

In one embodiment of the present invention, there are providedstructurally modified neurotoxins, wherein the first, second and/orthird regions of the structurally modified neurotoxins can be producedby recombinant DNA methodologies, i.e. produced recombinantly.

In another embodiment of the present invention, there are providedstructurally modified neurotoxins, wherein the first, second and/orthird region of the neurotoxin is isolated from a naturally existingClostridial neurotoxin.

Another embodiment of the present invention provides a modifiedneurotoxin comprising a botulinum toxin (such as a botulinum toxin typeA) which includes a structural modification which is effective to altera biological persistence of the modified neurotoxin relative to anidentical neurotoxin without the structural modification. The structuralmodification can comprise a deletion of amino acids 416 to 437 from alight chain of the neurotoxin (FIG. 3).

In still another embodiment of the present invention there is provided amodified neurotoxin (such as a botulinum toxin type A) which includes astructural modification which is effective to alter a biologicalpersistence of the modified neurotoxin relative to an identicalneurotoxin without the structural modification. The structuralmodification can comprise a deletion of amino acids 1 to 8 from a lightchain of the neurotoxin (FIG. 3).

Still further in accordance with the present invention there is provideda modified neurotoxin, such as a botulinum toxin type A, which includesa structural modification which is effective to alter a biologicalpersistence of the modified neurotoxin relative to an identicalneurotoxin without the structural modification. The structuralmodification can comprise a deletion of amino acids 1 to 8 and 416 to437 from a light chain of the neurotoxin (FIG. 3).

Still further in accordance with the present invention, there isprovided a modified botulinum toxin, such as a modified botulinum toxintype A, which includes a structural modification effective to alter abiological persistence of the modified neurotoxin relative to anidentical neurotoxin without said structural modification. Thestructural modification can comprise a substitution of leucine atposition 427 for an alanine and a substitution of leucine at position428 for an alanine in a light chain of said neurotoxin (FIG. 3).

Additionally, the scope of the present invention also includes methodsfor enhancing the biological persistence and/or or for enhancing thebiological activity of a neurotoxin. In these methods, a structuralmodification can be fused or added to the neurotoxin, for example, thestructural modification can be a biological persistence enhancingcomponent and/or a biological activity enhancing component. Examples ofstructural modifications that can be fused or added to the neurotoxinare a leucine-based motif, a tyrosine-based motif and an amino acidderivative. Examples of amino acid derivatives are a myristylated aminoacid, an N-glycosylated amino acid, and a phosphorylated amino acid. Anamino acid can be phosphorylated by, for example, protein kinase C,caseine kinase II or tyrosine kinase.

Also in accordance with the present invention, there are providedmethods for reducing the biological persistence and/or for reducing thebiological activity of a neurotoxin. These methods can comprise a stepof mutating an amino acid of the neurotoxin. For example, an amino acidof a leucine-based motif within the neurotoxin can be mutated. Also, forexample, one or more amino acids within a tyrosine-based motif of theneurotoxin can be mutated. Also, for example, an amino acid derivativefor DNA or amino acid sequence responsible for the derivatization of theamino acid can be mutated. The derivatized amino acid can be amyristylated amino acid, a N-glycosylated amino acid, or aphosphorylated amino acid. The phosphorylated amino acid can be producedby, for example, protein kinase C, caseine kinase II and tyrosinekinase. These mutations can be, for example, amino acid deletions oramino acids substitutions.

The present invention also includes methods for treating a condition.The methods can comprise a step of administering an effective dose of astructurally modified neurotoxin to a mammal to treat a condition. Thestructurally modified neurotoxin can include a structural modification.The structural modification is effective to alter the biologicalpersistence and/or the biological activity of the neurotoxin. Thesemethods for treating a condition can utilize a neurotoxin that does notcomprise a leucine-based motif. Also, these methods for treating acondition can utilize a neurotoxin, which includes a biologicalpersistence enhancing component and/or a biological activity enhancingcomponent. The biological persistence or activity enhancing componentcan comprise, for example, a tyrosine-based motif, a leucine-based motifor an amino acid derivative. The amino acid derivative can be, forexample, a myristylated amino acid, an N-glycosylated amino acid or aphosphorylated amino acid. The phosphorylated amino acid can be producedby, for example, protein kinase C, caseine kinase II or tyrosine kinase.The condition treated can be a neuromuscular disorder, an autonomicdisorder or pain. The treatment of a neuromuscular disorder can comprisea step of locally administering an effective amount of a modifiedneurotoxin to a muscle or a group of muscles. A method for treating anautonomic disorder can comprise a step of locally administering aneffective amount of a modified neurotoxin to a gland or glands. A methodfor treating pain can comprise a step of administering an effectiveamount of a modified neurotoxin to the site of the pain. In addition,the treatment of pain can comprise a step of administering an effectiveamount of a modified neurotoxin to the spinal cord.

Still further in accordance with the present invention, there areprovided methods for treating with modified neurotoxins conditionsincluding spasmodic dysphonia, laryngeal dystonia, oromandibulardysphonia, lingual dystonia, cervical dystonia, focal hand dystonia,blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebralpalsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus,limb spasticity, tics, tremors, bruxism, anal fissure, achalasia,dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessivegastrointestinal secretions, pain from muscle spasms, headache pain,brow furrows and skin wrinkles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows localization of GFP-botulinum toxin A light chain in (nervegrowth factor) NGF-differentiated live PC12 cells visualized on afluorescence inverted microscope.

FIG. 2 shows the localization of GFP-truncated botulinum toxin A lightchain in NGF-differentiated live PC12 cells visualized on a fluorescenceinverted microscope.

FIG. 3 shows the amino acid sequence for botulinum type A light chain.The amino acid sequence shown, minus the underlined amino acidsrepresents botulinum type A truncated light chain.

FIG. 4 shows the localization of GFP-botulinum toxin A light chain withLL to AA mutation at position 427 and 428 in NGF-differentiatedlive-PC12 cells visualized on a fluorescence inverted microscope.

FIG. 5 shows localization of fluorescently labeled anti-SNAP-25visualized in horizontal confocal sections ofstaurosporine-differentiated PC12 cells.

FIG. 6 shows an X-ray crystalographic structure of botulinum toxin typeA.

FIG. 7 shows localization of GFP-botulinum type B neurotoxin light chainin NGF-differentiated live PC12 cells visualized on a fluorescenceinverted microscope.

FIG. 8 shows sequence alignment and consensus sequence for botulinumtoxin type A HallA light chain and botulinum toxin type B Danish I lightchain.

FIG. 9 is a graph which illustrates the results of an in vitro ELISAassay carried out by the inventors demonstrating that a truncated LC/Ain vitro cleaves substrate at a slower rate or less efficiently thandoes non-truncated LC/A.

FIG. 10 shows a comparison of LC/A constructs expressed from E. coli forin vitro analysis.

DETAILED DESCRIPTION

The present invention is based upon the discovery that the biologicalpersistence and/or the biological activity of a neurotoxin can bealtered by structurally modifying the neurotoxin. In other words, amodified neurotoxin with an altered biological persistence and/orbiological activity can be formed from a neurotoxin containing orincluding a structural modification. In one embodiment, the structuralmodification includes the fusing of a biological persistence enhancingcomponent to the primary structure of a neurotoxin to enhance itsbiological persistence. In a preferred embodiment, the biologicalpersistence enhancing component is a leucine-based motif. Even morepreferably, the biological half-life and/or the biological activity ofthe modified neurotoxin is enhanced by about 100%. Generally speaking,the modified neurotoxin has a biological persistence of about 20% to300% more than an identical neurotoxin without the structuralmodification. That is, for example, the modified neurotoxin includingthe biological persistence enhancing component is able to cause asubstantial inhibition of neurotransmitter release for example,acetylcholine from a nerve terminal for about 20% to about 300% longerthan a neurotoxin that is not modified.

The present invention also includes within its scope a modifiedneurotoxin with a biological activity altered as compared to thebiological activity of the native or unmodified neurotoxin. For example,the modified neurotoxin can exhibit a reduced or an enhanced inhibitionof exocytosis (such as exocytosis of a neurotransmitter) from a targetcell with or without any alteration in the biological persistence of themodified neurotoxin.

In a broad embodiment of the present invention, a leucine-based motif isa run of seven amino acids. The run is organized into two groups. Thefirst five amino acids starting from the amino terminal of theleucine-based motif form a “quintet of amino acids.” The two amino acidsimmediately following the quintet of amino acids form a “duplet of aminoacids.” In a preferred embodiment, the duplet of amino acids is locatedat the carboxyl terminal region of the leucine-based motif. In anotherpreferred embodiment, the quintet of amino acids includes at 1 ast oneacidic amino acid selected from a group consisting of a glutamate and anaspartate.

The duplet of amino acid includes at least one hydrophobic amino acid,for example leucine, isoleucine, methionine, alanine, phenylalanine,tryptophan, valine or tyrosine. Preferably, the duplet of amino acid isa leucine-leucine, a leucine-isoleucine, an isoleucine-leucine or anisoleucine-isoleucine, leucine-methionine. Even more preferably, theduplet is a leucine-leucine.

In one embodiment, the leucine-based motif is xDxxxLL, wherein x can beany amino acids. In another embodiment, the leucine-based motif isxExxxLL, wherein E is glutamic acid. In another embodiment, the dupletof amino acids can include an isoleucine or a methionine, formingxDxxxLI or xDxxxLM, respectively. Additionally, the aspartic acid, D,can be replaced by a glutamic acid, E, to form xExxxLI, xExxxIL andxExxxLM. In a preferred embodiment, the leucine-based motif isphenylalanine-glutamate-phenylalanine-tyrosine-lysine-leucine-leucine,SEQ ID #1.

In another embodiment, the quintet of amino acids comprises at least onehydroxyl containing amino acid, for example, a serine, a threonine or atyrosine. Preferably, the hydroxyl containing amino acid can bephosphorylated. More preferably, the hydroxyl containing amino acid is aserine which can be phosphorylated to allow for the binding of adapterproteins.

Although non-modified amino acids are provided as examples, a modifiedamino acid is also contemplated to be within the scope of thisinvention. For example, leucine-based motif can include a halogenated,preferably, fluorinated leucine.

Various leucine-based motif are found in various species. A list ofpossible leucine-based motif derived from the various species that canbe used in accordance with this invention is shown in Table 1. This listis not intended to be limiting. TABLE 1 Species Sequence SEQID#Botulinum type A FEFYKLL 1 Rat VMAT1 EEKRAIL 2 Rat VMAT2 EEKMAIL 3 RatVAChT SERDVLL 4 Rat δ VDTQVLL 5 Mouse δ AEVQALL 6 Frog γ/δ SDKQNLL 7Chicken γ/δ SDRQNLI 8 Sheep δ ADTQVLM 9 Human CD3γ SDKQTLL 10 Human CD4SQIKRLL 11 Human δ ADTQALL 12 S. cerevisiae Vam3p NEQSPLL 13VMAT is vesicular monoamine transporter; VACht is vesicularacetylcholine transporter and S. cerevisiae Vam3p is a yeast homologueof synaptobrevin. Italicized serine residues are potential sites ofphosphorylation.

The modified neurotoxin can be formed from any neurotoxin. Also, themodified neurotoxin can be formed from a fragment of a neurotoxin, forexample, a botulinum toxin with a portion of the light chain and/orheavy chain removed. Preferably, the neurotoxin used is a Clostridialneurotoxin. A Clostridial neurotoxin comprises a polypeptide havingthree amino acid sequence regions. The first amino acid sequence regioncan include a target cell (i.e. a neuron) binding moiety which issubstantially completely derived from a neurotoxin selected from a groupconsisting of beratti toxin; butyricum toxin; tetanus toxin; botulinumtype A, B, C₁, D, E, F, and G. Preferably, the first amino acid sequenceregion is derived from the carboxyl terminal region of a toxin heavychain, H_(C). Also, the first amino acid sequence region can comprise atargeting moiety which can comprise a molecule (such as an amino acidsequence) that can bind to a receptor, such as a cell surface protein orother biological component on a target cell.

The second amino acid sequence region is effective to translocate thepolypeptide or a part thereof across an endosome membrane into thecytoplasm of a neuron. In one embodiment, the second amino acid sequenceregion of the polypeptide comprises an amine terminal of a heavy chain,H_(N), derived from a neurotoxin selected from a group consisting ofberatti toxin; butyricum toxin; tetanus toxin; botulinum type A, B, C₁,D, E, F, and G.

The third amino acid sequence region has therapeutic activity when it isreleased into the cytoplasm of a target cell, such as a neuron. In oneembodiment, the third amino acid sequence region of the polypeptidecomprises a toxin light chain, L, derived from a neurotoxin selectedfrom a group consisting of beratti toxin; butyricum toxin; tetanustoxin; botulinum type A, B, C₁, D, E, F, and G.

The Clostridial neurotoxin can be a hybrid neurotoxin. For example, eachof the neurotoxin's amino acid sequence regions can be derived from adifferent Clostridial neurotoxin serotype. For example, in oneembodiment, the polypeptide comprises a first amino acid sequence regionderived from the H_(C) of the tetanus toxin, a second amino acidsequence region derived from the H_(N) of botulinum type B, and a thirdamino acid sequence region derived from the light chain of botulinumserotype E. All other possible combinations are included within thescope of the present invention.

Alternatively, all three of the amino acid sequence regions of theClostridial neurotoxin can be from the same species and same serotype.If all three amino acid sequence regions of the neurotoxin are from thesame Clostridial neurotoxin species and serotype, the neurotoxin will bereferred to by the species and serotype name. For example, a neurotoxinpolypeptide can have its first, second and third amino acid sequenceregions derived from Botulinum type E. In which case, the neurotoxin isreferred as Botulinum type E.

Additionally, each of the three amino acid sequence regions can bemodified from the naturally occurring sequence from which they arederived. For example, the amino acid sequence region can have at leastone or more amino acids added or deleted as compared to the naturallyoccurring sequence.

A biological persistence enhancing component or a biological activityenhancing component, for example a leucine-based motif, can be fusedwith any of the above described neurotoxins to form a modifiedneurotoxin with an enhanced biological persistence and/or an enhancedbiological activity. “Fusing” as used in the context of this inventionincludes covalently adding to or covalently inserting in between aprimary structure of a neurotoxin. For example, a biological persistenceenhancing component and/or a biological activity enhancing component canbe added to a Clostridial neurotoxin which does not have a leucine-basedmotif in its primary structure. In one embodiment, a leucine-based motifis fused with a hybrid neurotoxin, wherein the third amino acid sequenceis derived from botulinum serotype A, B, C₁, C₂, D, E, F, or G. Inanother embodiment, the leucine-based motif is fused with a botulinumtype E.

In another embodiment, a biological persistence enhancing componentand/or a biological activity enhancing component is added to aneurotoxin by altering a cloned DNA sequence encoding the neurotoxin.For example, a DNA sequence encoding a biological persistence enhancingcomponent and/or a biological activity enhancing component is added to acloned DNA sequence encoding the neurotoxin into which the biologicalpersistence enhancing component and/or a biological activity enhancingcomponent is to be added. This can be done in a number of ways which arefamiliar to a molecular biologist of ordinary skill. For example, sitedirected mutagenesis or PCR cloning can be used to produce the desiredchange to the neurotoxin encoding DNA sequence. The DNA sequence canthen be reintroduced into a native host strain. In the case of botulinumtoxins the native host strain would be a Clostridium botulinum strain.Preferably, this host strain will be lacking the native botulinum toxingene. In an alternative method, the altered DNA can be introduced into aheterologous host system such as E. coli or other prokaryotes, yeast,insect cell lines or mammalian cell lines. Once the altered DNA has beenintroduced into its host, the recombinant toxin containing the addedbiological persistence enhancing component and/or a biological activityenhancing component can be produced by, for example, standardfermentation methodologies.

Similarly, a biological persistence enhancing component can be removedfrom a neurotoxin. For example, site directed mutagenesis can be used toeliminate biological persistence enhancing components, for example, aleucine-based motif.

Standard molecular biology techniques that can be used to accomplishthese and other genetic manipulations are found in Sambrook et al.(1989) which is incorporated in its entirety herein by reference.

In one embodiment, the leucine-based motif is fused with, or added to,the third amino acid sequence region of the neurotoxin. In a preferredembodiment, the leucine-based motif is fused with, or added to, theregion towards the carboxylic terminal of the third amino acid sequenceregion. More preferably, the leucine-based motif is fused with, or addedto, the carboxylic terminal of the third region of a neurotoxin. Evenmore preferably, the leucine-based motif is fused with, or added to thecarboxylic terminal of the third region of botulinum type E. The thirdamino acid sequence to which the leucine-based motif is fused or addedcan be a component of a hybrid or chimeric modified neurotoxin. Forexample, the leucine-based motif can be fused to or added to the thirdamino acid sequence region (or a part thereof) of one botulinum toxintype (i.e. a botulinum toxin type A), where the leucine-basedmotif-third amino acid sequence region has itself been fused to orconjugated to first and second amino acid sequence regions from anothertype (or types) of a botulinum toxin (such as botulinum toxin type Band/or E).

In another embodiment, a structural modification of a neurotoxin whichhas a pre-existing biological persistence enhancing component and/or abiological activity enhancing component, for example, a leucine-basedmotif includes deleting or substituting on or more amino acids of theleucine-bas d motif. In addition, a modified neurotoxin includes astructural modification which results in a neurotoxin with one or moreamino acids deleted or substituted in the leucine-based motif. Theremoval or substitution of one or more amino acids from the preexistingleucine-based motif is effective to reduce the biological persistenceand/or a biological activity of a modified neurotoxin. For example, thedeletion or substitution of one or more amino acids of the leucine-basedmotif of botulinum type A reduces the biological half-life and/or thebiological activity of the modified neurotoxin.

Amino acids that can be substituted for amino acids contained in abiological persistence enhancing component include alanine, aspargine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, tyrosine and othernaturally occurring amino acids as well as non-standard amino acids.

In the present invention the native botulinum type A light chain hasbeen shown to localize to differentiated PC12 cell membranes in acharacteristic pattern. Biological persistence enhancing components areshown to substantially contribute to this localization.

The data of the present invention demonstrates that when the botulinumtoxin type A light chain is truncated or when the leucine-bas d motif ismutated, the light chain substantially loses its ability to localize tothe membrane in its characteristic pattern. Localization to the cellularmembrane is believed to be a key factor in determining the biologicalpersistence and/or the biological activity of a botulinum toxin. This isbecause localization to a cell membrane can protect the localizedprotein from inter-cellular protein degrading.

FIGS. 1 and 2 show that deletion of the leucine-based motif from thelight chain of botulinum type A can change membrane localization of thetype A light chain. FIG. 1 shows localization of GFP-light chain Afusion protein in differentiated PC12 cells. The GFP fusion proteinswere produced and visualized in differentiated PC12 cells using methodswell known to those skilled in the art, for example, as described inGalli et al (1998) Mol Biol Cell 9:1437-1448, incorporated in itsentirety herein by reference; also, for example, as described inMartinez-Arca et al (2000) J Cell Biol 149:889-899, also incorporated inits entirety herein by reference. Localization of a GFP-truncated lightchain A is shown in FIG. 2. Comparing FIGS. 1 and 2, it can be seen thatthe pattern of localization is completely altered by the deletion of theN-terminus and C-terminus comprising the leucine-based motif. FIG. 3shows the amino acid sequence of the botulinum type A light chain. Theunderlined amino acid sequences indicate the amino acids that weredeleted in the truncated mutant. The leucine-based motif is indicated bythe asterisked bracket.

Further studies have been done in the present invention to analyze theeffect of specific amino acid substitutions within the leucine-bas dmotif. For example, in one study both leucine residues contained in theleucine-based motif were substituted for alanine residues. FIG. 4 showsthe fluorescent image of differentiated PC12 cells transfected with DNAencoding this di-leucine to di-alanine substituted GFP-botulinum A lightchain. As can be seen, the substitution of alanine for leucine atpositions 427 and 428 in the botulinum type A light chain substantiallychanges the localization characteristic of the light chain.

It is within the scope of this invention that a leucine-based motif, orany other persistence enhancing component and/or a biological activityenhancing component present on a light chain, can be used to protect theheavy chain as well. A random coil belt extends from the botulinum typeA translocation domain and encircles the light chain. It is possiblethat this belt keeps the two subunits in proximity to each other insidethe cell while the light chain is localized to the cell membrane. Thestructure of native botulinum toxin type A is shown in FIG. 6.

In addition, the data of the present invention shows that theleucine-based motif can be valuable in localizing the botulinum A toxinin close proximity to the SNAP-25 substrate within the cell. This canmean that the leucine-based motif is important not only for determiningthe half-life of the toxin but for determining the activity of the toxinas well. That is, the toxin will have a greater activity if it ismaintained in close proximity to the SNAP-25 substrate inside the cell.FIG. 5 shows the localization of SNAP-25 in horizontal confocal sectionsof differentiated PC12 cells (from Martinez-Arca et al (2000) J CellBiol 149:889-899). Similarity in the pattern of localization can be seenwhen comparing localization of botulinum type A light chain as seen inFIG. 1 to localization of SNAP-25 seen in FIG. 5.

The data of the present invention clearly shows that truncation of thelight chain, thereby deleting the leucine-based motif, or amino acidsubstitution within the leucine-based motif substantially changesmembrane localization of the botulinum type A light chain in nervecells. In both truncation and substitution a percentage of the alteredlight chain can localize to the cell membrane in a pattern unlike thatof the native type A light chain (see FIGS. 1, 2 and 4). This datasupports the presence of biological persistence enhancing componentsother than a leucine-based motif such as tyrosine motifs and amino acidderivatives. Use of these other biological persistence enhancingcomponents and/or a biological activity enhancing components in modifiedneurotoxins is also within the scope of the present invention.

Also within the scope of the present invention is more than onebiological persistence enhancing component used in combination in amodified neurotoxin to alter biological persistence of the neurotoxinthat is modified. The present invention also includes use of more thanone biological activity enhancing or biological activity reducingcomponents used in combination in a modified neurotoxin to alter thebiological activity of the neurotoxin that is modified.

Tyrosine-based motifs are within the scope of the present invention asbiological persistence and/or a biological activity altering components.Tyrosine-based motifs comprise the sequence Y-X-X-Hy where Y istyrosine, X is any amino acid and Hy is a hydrophobic amino acid.Tyrosine-based motifs can act in a manner that is similar to that ofleucine-based motifs. In FIG. 3 some of tyrosine motifs found in thetype A toxin light chain are bracketed. In addition, a tyrosine-basedmotif is found within the leucine-based motif which is indicated by anasterisked bracket in FIG. 3.

Also within the scope of the present invention are modified neurotoxinswhich comprise one or more biological persistence altering componentsand/or a biological activity enhancing components which occur naturallyin both botulinum toxin types A and B.

FIG. 7 shows localization of GFP-botulinum type B neurotoxin light chainin live, differentiated PC12 cells. Localization of the type B lightchain appears to be to an intracellular organelle. Similar localizationpattern is seen for GFP-truncated botulinum type A shown in FIG. 2.Localization of a botulinum toxin, or botulinum toxin light chain,within the cell is believed to be a key factor in determining biologicalpersistence and/or biological activity of the toxin. Therefore, thesedata appear to indicate that there are biological persistence alteringcomponent(s), and/or biological activity altering component(s), commonto the type A and type B botulinum toxins. These, and other biologicalpersistence altering components, and biological activity alteringcomponents, are contemplated for use in accordance with the presentinvention.

FIG. 8 shows a sequence alignment between type A and type B light chainsisolated from strains type A HallA (SEQ ID NO: 19) and type B Danish I(SEQ ID NO: 20) respectively. Light chains or heavy chains isolated fromother strains of botulinum toxin types A and B can also be used forsequence comparison. The shaded amino acids represent amino acididentities, or matches, between the chains. Each of the shaded aminoacids between amino acid position 10 and amino acid position 425 of theFIG. 8 consensus sequence, alone or in combination with any other shadedamino acid or amino acids, represents a biological persistence alteringcomponent that is within the scope of the present invention. Forexample, amino acids KAFK at positions 19 to 22, LNK at positions 304 to306, L at position 228 in combination with KL at positions 95 and 96,FDKLYK at positions 346 to 351, YL-T at positions 78 to 81, YYD atpositions 73 to 75 in combination with YL at positions 78 and 79 incombination with T a position 81, F at position 297 in combination withI at position 300 in combination with KL at positions 95 and 96 can bebiological persistence altering components for use within the scope ofthis invention. In addition, conserved regions of charge,hydrophobicity, hydrophilicity and/or conserved secondary, tertiary, orquaternary structures that may be independent of conserved sequence arewithin the scope of the present invention.

Amino acid derivatives are also within the scope of the presentinvention as biological persistence enhancing components and/or asbiological activity enhancing components. Examples of amino acidderivatives that act to effect biological persistence and/or biologicalactivity are phosphorylated amino acids. These amino acids include, forexample, amino acids phosphorylated by tyrosine kinase, protein kinase Cor casein kinase II. Other amino acid derivatives within the scope ofthe present invention as biological persistence enhancing componentsand/or as biological activity enhancing components are myristylatedamino acids and N-glycosylated amino acids.

In one broad aspect of the present invention, a method is provided fortreating a condition using a modified neurotoxin. The conditions caninclude, for example, skeletal muscle conditions, smooth muscleconditions, pain and glandular conditions. The modified neurotoxin canalso be used for cosmetics, for example, to treat brow furrows.

The neuromuscular disorders and conditions that can be treated with amodified neurotoxin include: for example, spasmodic dysphonia, laryngealdystonia, oromandibular and lingual dystonia, cervical dystonia, focalhand dystonia, blepharospasm, strabismus, hemifacial spasm, eyeliddisorders, spasmodic torticolis, cerebral palsy, focal spasticity andother voice disorders, spasmodic colitis, neurogenic bladder, anismus,limb spasticity, tics, tremors, bruxism, anal fissure, achalasia,dysphagia and other muscle tone disorders and other disorderscharacterized by involuntary movements of muscle groups can be treatedusing the present methods of administration. Other examples ofconditions that can be treated using the present methods andcompositions are lacrimation, hyperhydrosis, excessive salivation andexcessive gastrointestinal secretions, as well as other secretorydisorders. In addition, the present invention can be used to treatdermatological conditions, for example, reduction of brow furrows,reduction of skin wrinkles. The present invention can also be used inthe treatment of sports injuries.

Borodic U.S. Pat. No. 5,053,005 discloses methods for treating juvenilespinal curvature, i.e. scoliosis, using botulinum type A. The disclosureof Borodic is incorporated in its entirety herein by reference. In oneembodiment, using substantially similar methods as disclosed by Borodic,a modified neurotoxin can be administered to a mammal, preferably ahuman, to treat spinal curvature. In a preferred embodiment, a modifiedneurotoxin comprising botulinum type E fused with a leucine-based motifis administered. Even more preferably, a modified neurotoxin comprisingbotulinum type A-E with a leucine-bas d motif fused to the carboxylterminal of its light chain is administered to the mammal, preferably ahuman, to treat spinal curvature.

In addition, the modified neurotoxin can be administered to treat otherneuromuscular disorders using well known techniques that are commonlyperformed with botulinum type A. For example, the present invention canbe used to treat pain, for example, headache pain, pain from musclespasms and various forms of inflammatory pain. For example, Aoki U.S.Pat. No. 5,721,215 and Aoki U.S. Pat. No. 6,113,915 disclose methods ofusing botulinum toxin type A for treating pain. The disclosure of thesetwo patents is incorporated in its entirety herein by reference.

Autonomic nervous system disorders can also be treated with a modifiedneurotoxin. For example, glandular malfunctioning is an autonomicnervous system disorder. Glandular malfunctioning includes excessivesweating and excessive salivation. Respiratory malfunctioning is anotherexample of an autonomic nervous system disorder. Respiratorymalfunctioning includes chronic obstructive pulmonary disease andasthma. Sanders et al. disclose methods for treating the autonomicnervous system; for example, treating autonomic nervous system disorderssuch as excessive sweating, excessive salivation, asthma, etc., usingnaturally existing botulinum toxins. The disclosure of Sander et al. isincorporated in its entirety by reference herein. In one embodiment,substantially similar methods to that of Sanders et al. can be employed,but using a modified neurotoxin, to treat autonomic nervous systemdisorders such as the ones discussed above. For example, a modifiedneurotoxin can be locally applied to the nasal cavity of the mammal inan amount sufficient to degenerate cholinergic neurons of the autonomicnervous system that control the mucous secretion in the nasal cavity.

Pain that can be treated by a modified neurotoxin includes pain causedby muscle tension, or spasm, or pain that is not associated with musclespasm. For example, Binder in U.S. Pat. No. 5,714,468 discloses thatheadache caused by vascular disturbances, muscular tension, neuralgiaand neuropathy can be treated with a naturally occurring botulinumtoxin, for example Botulinum type A. The disclosures of Binder areincorporated in its entirety herein by reference. In one embodiment,substantially similar methods to that of Binder can be employed, butusing a modified neurotoxin, to treat headache, especially the onescaused by vascular disturbances, muscular tension, neuralgia andneuropathy. Pain caused by muscle spasm can also be treated by anadministration of a modified neurotoxin. For example, a botulinum type Efused with a leucine-based motif, preferably at the carboxyl terminal ofthe botulinum type E light chain, can be administered intramuscularly atthe pain/spasm location to alleviate pain.

Furthermore, a modified neurotoxin can be administered to a mammal totreat pain that is not associated with a muscular disorder, such asspasm. In one broad embodiment, methods of the present invention totreat non-spasm related pain include central administration orperipheral administration of the modified neurotoxin.

For example, Foster et al. in U.S. Pat. No. 5,989,545 discloses that abotulinum toxin conjugated with a targeting moiety can be administeredcentrally (intrathecally) to alleviate pain. The disclosures of Fosteret al. are incorporated in its entirety by reference herein. In oneembodiment, substantially similar methods to that of Foster et al. canbe employed, but using the modified neurotoxin according to thisinvention, to treat pain. The pain to be treated can be an acute pain,or preferably, chronic pain.

An acute or chronic pain that is not associated with a muscle spasm canalso be alleviated with a local, peripheral administration of themodified neurotoxin to an actual or a perceived pain location on themammal. In one embodiment, the modified neurotoxin is administeredsubcutaneously at or near the location of pain, for example, at or neara cut. In another embodiment, the modified neurotoxin is administeredintramuscularly at or near the location of pain, for example, at or neara bruise location on the mammal. In another embodiment, the modifiedneurotoxin is injected directly into a joint of a mammal, for treatingor alleviating pain caused by arthritic conditions. Also, frequentrepeated injection or infusion of the modified neurotoxin to aperipheral pain location is within the scope of the present invention.However, given the long lasting therapeutic effects of the presentinvention, frequent injection or infusion of the neurotoxin can not benecessary. For example, practice of the present invention can provide ananalgesic effect, per injection, for 2 months or longer, for example 27months, in humans.

Without wishing to limit the invention to any mechanism or theory ofoperation, it is believed that when the modified neurotoxin isadministered locally to a peripheral location, it inhibits the releaseof Neuro-substances, for example substance P, from the peripheralprimary sensory terminal by inhibiting SNARE-dependent exocytosis. Sincethe release of substance P by the peripheral primary sensory terminalcan cause or at least amplify pain transmission process, inhibition ofits release at the peripheral primary sensory terminal will dampen thetransmission of pain signals from reaching the brain.

In addition to having pharmacologic actions at the peripheral location,the modified neurotoxin of the present invention can also haveinhibitory effects in the central nervous system, upon directintrathecal administration, as set forth in U.S. Pat. No. 6,113,915, orupon peripheral administration, where presumably the modified toxin actsthrough retrograde transport via a primary sensory afferent. Thishypothesis of retrograde axonal transport is supported by published datawhich shows that botulinum type A can be retrograde transported to thedorsal horn when the neurotoxin is injected peripherally. Thus, work byWeigand et al, Nauny-Schmiedeberg's Arch. Pharmacol. 1976; 292, 161-165,and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol. 1974; 281, 47-56,showed that botulinum toxin is able to ascend to the spinal area byretrograde transport. As such, a modified neurotoxin, for examplebotulinum type A with one or more amino acids mutated from theleucine-based motif, injected at a peripheral location, for exampleintramuscularly, can be expected to be retrograde transported from theperipheral primary sensory terminal to a central region.

The amount of the modified neurotoxin administered can vary widelyaccording to the particular disorder being treated, its severity andother various patient variables including size, weight, age, andresponsiveness to therapy. Generally, the dose of modified neurotoxin tobe administered will vary with the age, presenting condition and weightof the mammal, preferably a human, to be treated. The potency of themodified neurotoxin will also be considered.

Assuming a potency (for a botulinum toxin type A) which is substantiallyequivalent to LD₅₀=2,730 U in a human patient and an average person is75 kg, a lethal dose (for a botulinum toxin type A) would be about 36U/kg of a modified neurotoxin. Therefore, when a modified neurotoxinwith such an LD₅₀ is administered, it would be appropriate to administerless than 36 U/kg of the modified neurotoxin into human subjects.Preferably, about 0.01 U/kg to 30 U/kg of the modified neurotoxin isadministered. More preferably, about 1 U/kg to about 15 U/kg of themodified neurotoxin is administered. Even more preferably, about 5 U/kgto about 10 U/kg modified neurotoxin is administered. Generally, themodified neurotoxin will be administered as a composition at a dosagethat is proportionally equivalent to about 2.5 cc/100 U. Those ofordinary skill in the art will know, or can readily ascertain, how toadjust these dosages for neurotoxin of greater or lesser potency. It isknown that botulinum toxin type B can be administered at a level aboutfifty times higher that that used for a botulinum toxin type A forsimilar therapeutic effect. Thus, the units amounts set forth above canbe multiplied by a factor of about fifty for a botulinum toxin type B.

Although examples of routes of administration and dosages are provided,the appropriate route of administration and dosage are generallydetermined on a case by case basis by the attending physician. Suchdeterminations are routine to one of ordinary skill in the art (see forexample, Harrison's Principles of Internal Medicine (1998), edited byAnthony Fauci et al., 14^(th) edition, published by McGraw Hill). Forexample, the route and dosage for administration of a modifiedneurotoxin according to the present disclosed invention can be selectedbased upon criteria such as the solubility characteristics of themodified neurotoxin chosen as well as the types of disorder beingtreated.

The modified neurotoxin can be produced by chemically linking theleucine-based motif to a neurotoxin using conventional chemical methodswell known in the art. For example, botulinum type E can be obtained byestablishing and growing cultures of Clostridium botulinum in afermenter, and then harvesting and purifying the fermented mixture inaccordance with known procedures.

The modified neurotoxin can also be produced by recombinant techniques.Recombinant techniques are preferable for producing a neurotoxin havingamino acid sequence regions from different Clostridial species or havingmodified amino acid sequence regions. Also, the recombinant technique ispreferable in producing botulinum type A with the leucine-based motifbeing modified by deletion. The technique includes steps of obtaininggenetic materials from natural sources, or synthetic sources, which havecodes for a cellular binding moiety, an amino acid sequence effective totranslocate the neurotoxin or a part thereof, and an amino acid sequencehaving therapeutic activity when released into a cytoplasm of a targetcell, preferably a neuron. In a preferred embodiment, the geneticmaterials have codes for the biological persistence enhancing component,preferably the leucine-based motif, the H_(C), the H_(N) and the lightchain of the Clostridial neurotoxins and fragments thereof. The geneticconstructs are incorporated into host cells for amplification by firstfusing the genetic constructs with a cloning vectors, such as phages orplasmids. Then the cloning vectors are inserted into a host, forexample, Clostridium sp., E. coli or other prokaryotes, yeast, insectcell lin or mammalian cell lines. Following the expressions of therecombinant genes in host cells, the resultant proteins can be isolatedusing conventional techniques.

There are many advantages to producing these modified neurotoxinsrecombinantly. For example, to form a modified neurotoxin, a modifyingfragment, or component must be attached or inserted into a neurotoxin.The production of neurotoxin from anaerobic Clostridium cultures is acumbersome and time-consuming process including a multi-steppurification protocol involving several protein precipitation steps andeither prolonged and repeated crystallization of the toxin or severalstages of column chromatography. Significantly, the high toxicity of theproduct dictates that the procedure must be performed under strictcontainment (BL-3). During the fermentation process, the foldedsingle-chain neurotoxins are activated by endogenous Clostridialproteases through a process termed nicking to create a dichain.Sometimes, the process of nicking involves the removal of approximately10 amino acid residues from the single-chain to create the dichain formin which the two chains remain covalently linked through the intrachaindisulfide bond.

The nicked neurotoxin is much more active than the unnicked form. Theamount and precise location of nicking varies with the serotypes of thebacteria producing the toxin. The differences in single-chain neurotoxinactivation and, hence, the yield of nicked toxin, are due to variationsin the serotype and amounts of proteolytic activity produced by a givenstrain. For example, greater than 99% of Clostridial botulinum serotypeA single-chain neurotoxin is activated by the Hall A Clostridialbotulinum strain, whereas serotype B and E strains produce toxins withlower amounts of activation (0 to 75% depending upon the fermentationtime). Thus, the high toxicity of the mature neurotoxin plays a majorpart in the commercial manufacture of neurotoxins as therapeutic agents.

The degree of activation of engineered Clostridial toxins is, therefore,an important consideration for manufacture of these materials. It wouldbe a major advantage if neurotoxins such as botulinum toxin and tetanustoxin could be expressed, recombinantly, in high yield inrapidly-growing bacteria (such as heterologous E. coli cells) asrelatively non-toxic single-chains (or single chains having reducedtoxic activity) which are safe, easy to isolate and simple to convert tothe fully-active form.

With safety being a prime concern, previous work has concentrated on theexpression in E. coli and purification of individual H and light chainsof tetanus and botulinum toxins; these isolated chains are, bythemselves, non-toxic; see Li t al., Biochemistry 33:7014-7020 (1994);Zhou et al., Biochemistry 34:15175-15181 (1995), hereby incorporated byreference herein. Following the separate production of these peptidechains and under strictly controlled conditions the H and light chainscan be combined by oxidative disulphide linkage to form theneuroparalytic di-chains.

EXAMPLES

The following non-limiting examples provide those of ordinary skill inthe art with specific preferred methods to treat non-spasm related painwithin the scope of the present invention and are not intended to limitthe scope of the invention.

Example 1 Treatment of Pain Associated with Muscle Disorder

An unfortunate 36 year old woman has a 15 year history oftemporomandibular joint disease and chronic pain along the masseter andtemporalis muscles. Fifteen years prior to evaluation she notedincreased immobility of the jaw associated with pain and jaw opening andclosing and tenderness along each side of her face. The left side isoriginally thought to be worse than the right. She is diagnosed ashaving temporomandibular joint (TMJ) dysfunction with subluxation of thejoint and is treated with surgical orthoplasty meniscusectomy andcondyle resection.

She continues to have difficulty with opening and closing her jaw afterthe surgical procedures and for this reason, several years later, asurgical procedure to replace prosthetic joints on both sides isperformed. After the surgical procedure progressive spasms and deviationof the jaw ensues. Further surgical revision is performed subsequent tothe original operation to correct prosthetic joint loosening. The jawcontinues to exhibit considerable pain and immobility after thesesurgical procedures. The TMJ remained tender as well as the muscleitself. There are tender points over the temporomandibular joint as wellas increased tone in the entire muscle. She is diagnosed as havingpost-surgical myofascial pain syndrome and is injected with the modifiedneurotoxin into the masseter and temporalis muscles; the modifiedneurotoxin is botulinum type E comprising a leucine-based motif. Theparticular dose as well as the frequency of administrations depends upona variety of factors within the skill of the treating physician.

Several days after the injections she noted substantial improvement inher pain and reports that her jaw feels looser. This gradually improvesover a 2 to 3 week period in which she notes increased ability to openthe jaw and diminishing pain. The patient states that the pain is betterthan at any time in the last 4 years. The improved condition persistsfor up to 27 months after the original injection of the modifiedneurotoxin.

Example 2 Treatment of Pain Subsequent to Spinal Cord Injury

A patient, age 39, experiencing pain subsequent to spinal cord injury istreated by intrathecal administration, for example, by spinal tap or bycatherization (for infusion) to the spinal cord, with the modifiedneurotoxin; the modified neurotoxin is botulinum type E comprising aleucine-based motif. The particular toxin dose and site of injection, aswell as the frequency of toxin administrations, depend upon a variety offactors within the skill of the treating physician, as previously setforth. Within about 1 to about 7 days after the modified neurotoxinadministration, the patient's pain is substantially reduced. The painalleviation persists for up to 27 months.

Example 3 Peripheral Administration of a Modified Neurotoxin to Treat“Shoulder-Hand Syndrome”

Pain in the shoulder, arm, and hand can develop, with musculardystrophy, osteoporosis and fixation of joints. While most common aftercoronary insufficiency, this syndrome can occur with cervicalosteoarthritis or localized shoulder disease, or after any prolongedillness that requires the patient to remain in bed.

A 46 year old woman presents a shoulder-hand syndrome type pain. Thepain is particularly localized at the deltoid region. The patient istreated by a bolus injection of a modified neurotoxin subcutaneously tothe shoulder; preferably the modified neurotoxin is botulinum type Ecomprising a leucine-based motif. The modified neurotoxin can also be,for example, modified botulinum type A, B, C1, C2, D, E, F or G whichcomprise a leucine-based motif. The particular dose as well as thefrequency of administrations depends upon a variety of factors withinthe skill of the treating physician, as previously set forth. Within 1-7days after modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 4 Peripheral Administration of a Modified Neurotoxin to TreatPostherapeutic Neuralgia

Postherapeutic neuralgia is one of the most intractable of chronic painproblems. Patients suffering this excruciatingly painful process oftenare elderly, have debilitating disease, and are not suitable for majorinterventional procedures. The diagnosis is readily made by theappearance of the healed lesions of herpes and by the patient's history.The pain is intense and emotionally distressing. Postherapeuticneuralgia can occur anywhere, but is most often in the thorax.

A 76 year old man presents a postherapeutic type pain. The pain islocalized to the abdomen region. The patient is treated by a bolusinjection of a modified neurotoxin intradermally to the abdomen; themodified neurotoxin is, for example, botulinum type A, B, C1, C2, D, E,F and/or G. The modified neurotoxin comprises a leucine-based motifand/or additional tyrosine-based motifs. The particular dose as well asthe frequency of administration depends upon a variety of factors withinthe skill of the treating physician, as previously set forth. Within 1-7days after modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 5 Peripheral Administration of a Modified Neurotoxin to TreatNasopharyngeal Tumor Pain

These tumors, most often squamous cell carcinomas, are usually in thefossa of Rosenmuller and can invade the base of the skull. Pain in theface is common. It is constant, dull-aching in nature.

A 35 year old man presents a nasopharyngeal tumor type pain. Pain isfound at the lower left cheek. The patient is treated by a bolusinjection of a modified neurotoxin intramuscularly to the cheek,preferably the modified neurotoxin is botulinum type A, B, C1, C2, D, E,F or G comprising additional biological persistence enhancing amino acidderivatives, for example, tyrosine phosphorylations. The particular doseas well as the frequency of administrations depends upon a variety offactors within the skill of the treating physician. Within 1-7 days aftr modified neurotoxin administration the patient's pain is substantiallyalleviated. The duration of the pain alleviation is from about 7 toabout 27 months.

Example 6 Peripheral Administration of a Modified Neurotoxin to TreatInflammatory Pain

A patient, age 45, presents an inflammatory pain in the chest region.The patient is treated by a bolus injection of a modified neurotoxinintramuscularly to the chest, preferably the modified neurotoxin isbotulinum type A, B, C1, C2, D, E, F or G comprising additionaltyrosine-based motifs. The particular dose as well as the frequency ofadministrations depends upon a variety of factors within the skill ofthe treating physician, as previously set forth. Within 1-7 days aftermodified neurotoxin administration the patient's pain is substantiallyalleviated. The duration of the pain alleviation is from about 7 toabout 27 months.

Example 7 Treatment of Excessive Sweating

A male, age 65, with excessive unilateral sweating is treated byadministering a modified neurotoxin. The dose and frequency ofadministration depends upon degree of desired effect. Preferably, themodified neurotoxin is botulinum type A, B, C1, C2, D, E, F and/or G.The modified neurotoxins comprise a leucine-based motif. Theadministration is to the gland nerve plexus, ganglion, spinal cord orcentral nervous system. The specific site of administration is to bedetermined by the physician's knowledge of the anatomy and physiology ofthe target glands and secretory cells. In addition, the appropriatespinal cord level or brain area can be injected with the toxin. Thecessation of excessive sweating after the modified neurotoxin treatmentis up to 27 months.

Example 8 Post Surgical Treatments

A female, age 22, presents a torn shoulder tendon and undergoesorthopedic surgery to repair the tendon. After the surgery, the patientis administered intramuscularly with a modified neurotoxin to theshoulder. The modified neurotoxin can botulinum type A, B, C, D, E, F,and/or G wherein one or more amino acids of a biological persistenceenhancing component are deleted from the toxin. For example, one or moreleucine residues can be deleted from and/or mutated from theleucine-based motif in botulinum toxin serotype A. Alternatively, one ormore amino acids of the leucine-based motif can be substituted for otheramino acids. For example, the two leucines in the leucine-based motifcan be substituted for alanines. The particular dose as well as thefrequency of administrations depends upon a variety of factors withinthe skill of the treating physician. The specific site of administrationis to be determined by the physician's knowledge of the anatomy andphysiology of the muscles. The administered modified neurotoxin reducesmovement of the arm to facilitate the recovery from the surgery. Theeffect of the modified neurotoxin is for about five weeks or less.

Example 9 Cloning, Expression and Purification of the BotulinumNeurotoxin Light Chain Gene

This example describes methods to clone and express a DNA nucleotidesequence encoding a botulinum toxin light chain and purify the resultingprotein product. A DNA sequence encoding the botulinum toxin light chaincan be amplified by PCR protocols which employ syntheticoligonucleotides having sequences corresponding to the 5′ and 3′ endregions of the light chain gene. Design of the primers can allow for theintroduction of restriction sites, for example, Stu I and EcoR Irestriction sites into the 5′ and 3′ ends of the botulinum toxin lightchain gene PCR product. These restriction sites can be subsequently usedto facilitate unidirectional subcloning of the amplification products.Additionally, these primers can introduce a stop codon at the C-terminusof the light chain coding sequence. Chromosomal DNA from C. botulinum,for example, strain HallA, can serve as a template in the amplificationreaction.

The PCR amplification can be performed in a 0.1 mL volume containing 10mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of eachdeoxynucleotide triphosphate (dNTP), 50 pmol of each primer, 200<ng ofgenomic DNA and 2.5 units of Taq DNA polymerase. The reaction mixturecan be subjected to 35 cycles of denaturation (1 minute at 94° C.),annealing (2 minutes at 55° C.) and polymerization (2 minutes at 72°C.). Finally, the reaction can be extended for an additional minutes at72° C.

The PCR amplification product can be digested with for example, Stu Iand EcoR I, to release the light chain encoding, cloned, PCR DNAfragment. This fragment can then be purified by, for example, agarosegel electrophoresis, and ligated into, for example, a Sma I and EcoR Idigested pBluescript II SK phagemid. Bacterial transformants, forexample, E coli, harboring this recombinant phagemid can be identifiedby standard procedures, such as blue/white screening. Clones comprisingthe light chain encoding DNA can be identified by DNA sequence analysisperformed by standard methods. The cloned sequences can be confirmed bycomparing the cloned sequences to published sequences for botulinumlight chains, for example, Binz, et al., in J. Biol. Chem. 265, 9153(1990), Thompson et al., in Eur. J. Biochem. 189, 73 (1990) and Minton,Clostridial Neurotoxins, The Molecular Pathogenesis of Tetanus andBotulism p. 161-191, Edited by C. Motecucco (1995).

The light chain can be subcloned into an expression vector, for example,pMal-P2. pMal-P2 harbors the malE gene encoding MBP (maltose bindingprotein) which is controlled by a strongly inducible promoter, P_(taC).

To verify expression of the botulinum toxin light chain, a well isolatedbacterial colony harboring the light chain gene containing pMal-P2 canbe used to inoculate L-broth containing 0.1 mg/ml ampicillin and 2%(w/v) glucose, and grown overnight with shaking at 30° C. The overnightcultures can be diluted 1:10 into fresh L-broth containing 0.1 mg/ml ofampicillin and incubated for 2 hours. Fusion protein expression can beinduced by addition of IPTG to a final concentration of 0.1 mM. After anadditional 4 hour incubation at 30° C., bacteria can be collected bycentrifugation at 6,000×g for 10 minutes.

A small-scale SDS-PAGE analysis can confirm the presence of a 90 kDaprotein band in samples derived from IPTG-induced bacteria. This MWwould be consistent with the predicted size of a fusion protein havingMBP (˜40 kDa) and botulinum toxin light chain (˜50 kDa) components.

The presence of the desired fusion proteins in IPTG-induced bacterialextracts can be confirmed by western blotting using the polyclonalanti-L chain probe described by Cenci di Bello et al., in Eur. J.Biochem. 219, 161 (1993). Reactive bands on PVDF membranes (Pharmacia;Milton Keynes, UK) can be visualized using an anti-rabbit immunoglobulinconjugated to horseradish peroxidase (BioRad; Hemel Hempstead, UK) andthe ECL detection system (Amersham, UK). Western blotting resultstypically confirm the presence of the dominant fusion protein togetherwith several faint bands corresponding to proteins of lower MW than thefully sized fusion protein. This observation suggests that limited dgradation of the fusion protein occurred in the bacteria or during theisolation procedure.

To produce the subcloned light chain, pellets from 1 liter cultures ofbacteria expressing the wild-type Botulinum neurotoxin light chainproteins can be resuspended in column buffer [10 mM Tris-HCl (pH 8.0),200 mM NaCl, 1 mM EGTA and 1 mM DTT] containing 1 mMphenylmethanesulfonyl fluoride (PMSF) and 10 mM benzamidine, and lysedby sonication. The lysates can be cleared by centrifugation at 15,000×gfor 15 minutes at 4° C. Supernatants can be applied to an amyloseaffinity column [2>10 cm, 30 ml resin] (New England BioLabs; Hitchin,UK). Unbound proteins can be washed from the resin with column bufferuntil the eluate is free of protein as judged by a stable absorbancereading at 280 nm. The bound MBP-L chain fusion protein can besubsequently eluted with column buffer containing 10 mM maltose.Fractions containing the fusion protein can be pooled and dialyzedagainst 20 mM Tris-HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM,CaCl₂ and 1 mM DTT for 72 hours at 4° C.

The MBP-L chain fusion proteins can be purified after release from thehost bacteria. Release from the bacteria can be accomplished byenzymatically degrading or mechanically disrupting the bacterial cellmembrane. Amylose affinity chromatography can be used for purification.Recombinant wild-type or mutant light chains can be separated from thesugar binding domains of the fusion proteins by site-specific cleavagewith Factor Xa. This cleavage procedure typically yields free MBP, freelight chains and a small amount of uncleaved fusion protein. While theresulting light chains present in such mixtures can be shown to possessthe desired activities, an additional purification step can be employed.For example, the mixture of cleavage products can be applied to a secondamylose affinity column which binds both the MBP and uncleaved fusionprotein. Free light chains can be isolated in the flow through fraction.

Example 10 Reconstitution of Native Light Chain, Recombinant Wild-TypeLight Chain with Purified Heavy Chain

Native heavy and light chains can be dissociated from a BoNT with 2 Murea, reduced with 100 mM DTT and then purified according to establishedchromatographic procedures. For example, Kozaki et al. (1981, Japan J.Med. Sci. Biol. 34, 61) and Maisey et al. (1988, Eur. J. Biochem. 177,683). A purified heavy chain can be combined with an equimolar amount ofeither native light chain or a recombinant light chain. Reconstitutioncan be carried out by dialyzing the samples against a buffer consistingof 25 mM Tris (pH 8.0), 50 μM zinc acetate and 150 mM NaCl over 4 daysat 4° C. Following dialysis, the association of the recombinant lightchain and native heavy chain to form disulfide linked 150 kDa dichainsis monitored by SDS-PAGE and/or quantified by densitometric scanning.

Example 11 Production of a Modified Neurotoxin with an EnhancedBiological Persistence

A modified neurotoxin can be produced by employing recombinanttechniques in conjunction with conventional chemical techniques.

A neurotoxin chain, for example a botulinum light chain that is to befused with a biological persistence enhancing component to form amodified neurotoxin can be produced recombinantly and purified asdescribed in example 9.

The recombinant neurotoxin chain derived from the recombinant techniquescan be covalently fused with (or coupled to) a biological persistenceenhancing component, for example a leucine-based motif, a tyrosine-basedmotif and/or an amino acid derivative. Peptide sequences comprisingbiological persistence enhancing components can be synthesized bystandard t-Boc/Fmoc technologies in solution or solid phase as is knownto those skilled in the art. Similar synthesis techniques are alsocovered by the scope of this invention, for example, methodologiesemployed in Milton et al. (1992, Biochemistry 31, 8799-8809) and Swainet al. (1993, Peptide Research 6, 147-154). One or more synthesizedbiological persistence enhancing components can be fused to the lightchain of botulinum type A, B, C1, C2, D, E, F or G at, for example, thecarboxyl terminal end of the toxin. The fusion of the biologicalpersistence enhancing components is achieved by chemical coupling usingreagents and techniques known to those skilled in the art, for examplePDPH/EDAC and Traut's reagent chemistry.

Alternatively, a modified neurotoxin can be produced recombinantlywithout the step of fusing the biological persistence enhancingcomponent to a recombinant botulinum toxin chain. For example, arecombinant neurotoxin chain, for example, a botulinum light chain,derived from the recombinant techniques of example 9 can be producedwith a biological persistence enhancing component, for example aleucine-based motif, a tyrosine-based motif and/or an amino acidderivative. For example, one or more DNA sequences encoding biologicalpersistence enhancing components can be added to the DNA sequenceencoding the light chain of botulinum type A, B, C1, C2, D, E, F or G.This addition can be done by any number of methods used for sitedirected mutagenesis which are familiar to those skilled in the art.

The recombinant modified light chain containing the fused or addedbiological persistence enhancing component can be reconstituted with aheavy chain of a neurotoxin by the method described in example 10thereby producing a complete modified neurotoxin.

The modified neurotoxins produced according to this example have anenhanced biological persistence. Preferably, the biological persist nceis enhanced by about 20% to about 300% relative to an identicalneurotoxin without the additional biological persistence enhancingcomponents( ).

Example 12 Production of a Modified Neurotoxin with a Reduced BiologicalPersistence

A modified neurotoxin with a reduced biological persistence can beproduced by employing recombinant techniques. For example, a botulinumlight chain derived from the recombinant techniques of example 9 can beproduced without a biological persistence enhancing component. Forexample, one or more leucine-based motifs, tyrosine-based motifs and/oramino acid derivatives can be mutated. For example, one or more DNAsequences encoding biological persistence enhancing components can beremoved from the DNA sequence encoding the light chain of botulinum typeA, B, C1, C2, D, E, F or G. For example, the DNA sequence encoding theleucine based motif can be removed from the DNA sequence encodingbotulinum type A light chain. Removal of the DNA sequences can be doneby any number of methods familiar to those skilled in the art.

The recombinant modified light chain with the deleted biologicalpersistence enhancing component can be reconstituted with a heavy chainof a neurotoxin by the method described in example 10 thereby producinga complete modified neurotoxin.

The modified neurotoxin produced according to this example has a reducedbiological persistence. Preferably, the biological persistence isreduced by about 20% to about 300% relative to an identical neurotoxin,for example botulinum type A, with the leucine-based motif.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of modified neurotoxins can be effectivelyused in the methods of the present invention in place of Clostridialneurotoxins. Also, the corresponding genetic codes, i.e. DNA sequence,to the modified neurotoxins are also considered to be part of thisinvention. Additionally, the present invention includes peripheraladministration methods wherein two or more modified neurotoxins, forexample botulinum type E with a fused leucine-based motif and botulinumtype B comprising a leucine-based motif, are administered concurrentlyor consecutively. While this invention has been described with respectto various specific examples and embodiments, it is to be understoodthat the invention is not limited thereto and that it can be variouslypracticed with the scope of the following claims.

Example 13 Production of a Modified Neurotoxin with a Reduced BiologicalPersistence

Localization to the cellular membrane is likely a key factor indetermining the biological persistence of botulinum toxins. This isbecause localization to a cell membrane can protect the localizedprotein from inter-cellular protein degrading complexes.

It is well known and generally accepted that the biological persistenceof botulinum type B neurotoxin is shorter than the biologicalpersistence of botulinum type A neurotoxin. In this work, it wasdemonstrated that when the botulinum toxin type A light chain istruncated, which comprises removing the leucine-based motif, the lightchain substantially loses its ability to localize to the cellularmembrane in its characteristic pattern. In fact, truncated type A lightchain localizes to the cellular membrane in a pattern similar to that ofbotulinum toxin type B light chain.

Therefore, it can be hypothesized that truncated botulinum type A has areduced biological persistence and/or a reduced biological activitysimilar to that of botulinum toxin type B.

Example 14 Production of a Modified Neurotoxin with an AlteredBiological Persistence

Localization to the cellular membrane is likely a key factor indetermining the biological persistence of botulinum toxins. This isbecause localization to a cell membrane can protect the localizedprotein from inter-cellular protein degrading complexes.

In this work, it was demonstrated that when the botulinum toxin type Alight chain is mutated, changing the two leucines at positions 427 and428 to alanines (FIG. 3), the light chain substantially loses itsability to localize to the cellular membrane in its characteristicpattern.

From this data it can be concluded that the mutated botulinum type A hasan altered biological persistence.

Example 15 In Vitro Cleavage of SNAP 25 by Truncated LC/A

As illustrated by FIG. 9, an in vitro ELISA assay was carried out by theinventors demonstrating that a truncated LC/A in vitro cleaves SNAP-25substrate less efficiently than does non-truncated LC/A. The datadisplayed is not a measure of inhibition of exocytosis but a measure ofthe in vitro formation of SNAP-25 cleavage. The assay was carried out asfollows:

Materials:

BirA-SNAP25₁₂₈₋₂₀₆—this is a recombinant substrate for LC/A, consistingof a BirA signal sequence fused to the N-terminus of residues 128-206 ofSNAP25. This fusion construct was produced in E. coli and the BirAsignal sequence was biotinylated by the E. coli. Microtiter plates werecoated with streptavidin. The toxin used was BoNT/A complex or LC/Aconstructs. The primary antibody was anti-SNAP25₁₉₇ antibody. Thisantibody recognizes the C-terminus of SNAP25 following cleavage by Typ Atoxin (BirA-SNAP25₁₂₈₋₁₉₇). The secondary antibody was goat, anti-rabbitIgG conjugated to horseradish peroxidase. The ImmunoPure TMB substratewas from Pierce, a calorimetric substrate for horseradish peroxidase.The antibody that recognizes the cleaved product SNAP25₁₉₇ is specificfor that cleaved product and does not recognize the full lengthuncleaved substrate SNAP25₂₀₆.

Method:

BirA-SNAP25₁₂₈₋₂₀₆ was bound to streptavidin on a microtiter plate. Tothe plates were added serial dilutions of BoNT/A 900 kDa complex,His6-S-nativeLC/A, or His6-S-truncLC/A-His6. All toxin samples werepre-incubated with DTT (this is not required for the LC/A constructs,but they were treated the same as the BoNT/A complex). The toxin sampleswere incubated with the substrate for 90 minutes at 37° C. The toxin wasremoved and the bound substrate was incubated with anti-SNAP25₁₉₇antibody. Unbound antibody was washed away and the plates were thenincubated with the secondary antibody (anti-rabbit IgG conjugated tohorseradish peroxidase). Unbound antibody was again washed away and acalorimetric assay for horseradish peroxidase was performed. The assaywas quantified by reading the absorbance at 450 nm.

In other work by the inventors disclosed herein the light chainconstructs that were expressed in the PC-12 cells were express ddirectly in the PC-12 cells and do not contain any tags. The light chainconstructs that have been expressed from E. coli for these in vitroassays contain affinity tags for purification purposes (these tags arenot present on the prot ins expressed in the PC-12 cells, as disclosedherein). The LC/A expressed in PC12 was the fusion protein GFP-LC/A.Between the GFP and the LC/A there is a set of Gly to separate bothproteins.

An explanation of the various constructs follows: Complex (red in thegraph) this is BoNT/A 900 kDa complex isolated from C. botulinumTruncated LC/A—construct lacking 8 amino acids at the N-terminus and 22amino acids at the C-terminus. However, this construct does contain a6-histidine and an S-tag at the N-terminus as well as a 6-histidine tagat the C-terminus.

Dialyzed Truncated LC/A—same as Truncated LC/A, but imidazole resultingfrom the purification has been removed.

Full LC/A (Dark green in graph)—native LC/A construct (full-length), butcontaining the N-terminal 6-histidine and S-tag. Does not have theC-terminal 6-histidine.

Dialyzed Full LC/A (Light green in graph)—Same as Full LC/A, butimidazole resulting from the purification has been removed.

To graphically depict these differences, FIG. 10 shows the veryN-terminus and the very C-terminus of these constructs (the middleportion of the LC/A proteins is not shown). What is referred to asWildtype corresponds to the native LC/A that the inventors had expresseddirectly in the PC-12 cells (this is construct that the inventorsdemonstrated activity with via Western blot analysis of the cleavedSNAP25 product). Truncated LC/A is the truncated light chain containingthe His and S-tags. N-His-LC/A is what was referred to as Full LC/A inFIG. 9.

1-68. (canceled)
 69. A modified neurotoxin comprising: a neurotoxinincluding a structural modification, wherein said structuralmodification is effective to enhance a biological persistence of saidmodified neurotoxin relative to an identical neurotoxin without saidstructural modification, and wherein said modified neurotoxin isstructurally different from a naturally existing neurotoxin.
 70. Themodified neurotoxin of claim 1 wherein said structural modificationcomprises 1 to about 22 amino acids.
 71. The modified neurotoxin ofclaim 1 wherein said structural modification comprises an amino acid,said amino acid comprising an R group of 1 to about 12 carbon atoms. 72.The modified neurotoxin of claim 1 wherein said structural modificationcomprises a leucine-based motif (SEQ ID NO: 1).
 73. The modifiedneurotoxin of claim 1 wherein said structural modification comprises atyrosine-based motif.
 74. The modified neurotoxin of claim 1 whereinsaid structural modification comprises an amino acid sequence of abotulinum type A light chain and of an amino acid sequence of a type Blight chain.
 75. The modified neurotoxin of claim 74 wherein saidstructural modification comprises the amino acid sequence KAFK.
 76. Themodified neurotoxin of claim 74 wherein said structural modificationcomprises the amino acid sequence YYD in combination with the amino acidsequence YYL in combination with the amino acid sequence T.
 77. Themodified neurotoxin of claim 1 wherein said structural modificationcomprises an amino acid derivative.
 78. The modified neurotoxin of claim1 wherein said neurotoxin is selected from the group consisting ofbotulinum toxin type A, B, C₁, C₂, D, E, F and G.
 79. The modifiedneurotoxin of claim 1 wherein said neurotoxin is botulinum toxin type A.